The goal of this project is to determine how cellular localization of the V(D)J recombinase is regulated during genotoxic stress, and if this correlates with reduced DNA cleavage activity. Central to lymphocyte development is the assembly of immunoglobulin and T cell receptor genes from component gene segments. In this process, DNA double strand breaks (DSBs) are generated in an intermediate step by the V(D)J recombinase, which consists of RAG1 and RAG2. This is a particularly risky step, as DSBs are the most genotoxic form of DNA damage. In accordance with this, many lymphomas and leukemias contain characteristic chromosomal translocations between an oncogene and an antigen receptor locus, with breakpoints in the latter locus often attributed to RAG-mediated cleavage. Lymphocytes undergoing genotoxic stress would be at a greater risk of forming such deleterious chromosomal translocations, as more DNA ends would be available to aberrantly join with RAG-produced breaks. Furthermore, DNA damage induces cell cycle arrest in G1. However, as the V(D)J recombinase is stabilized in G1, prolonging this cell cycle phase would be expected to increase the probability of aberrant recombination events. In our preliminary studies using fluorescently tagged RAG proteins, we determined that DNA damage induced by ionizing radiation (IR) prompted a re-localization of the V(D)J recombinase in the cell, which in effect sequestered the enzyme complex from the genome. This was a transient effect, as the pre-DNA damage co-localization properties of the V(D)J recombinase were re- established upon DNA repair. Moreover, the IR-induced redistribution of the V(D)J recombinase was blocked by addition of an inhibitor to DNA damage response (DDR) kinases. Given these results, we hypothesize that during genotoxic stress, the DDR system sequesters the V(D)J recombinase from the genome by disrupting interactions between RAG2 and Histone H3 trimethylated on Lysine 4 (H3K4me3). In this project, we will use fluorescence microscopy combined with assays to monitor cellular protein-protein and protein-DNA interactions. Three specific aims are proposed. First, we will ascertain if spatial regulation of the V(D)J recombinase is a ubiquitous process independent of cell type. Second, we will determine if the interaction between RAG2 and H3K4me3-bound chromatin is disrupted upon IR exposure, and if this occurs independently of RAG1. In addition, the specific DDR factor that mediates this effect will be identified. Third, we will determine if the sequence-specific association with and cleavage activity on RAG recognition sites in the antigen receptor loci are affected by IR-induced DNA damage. This project will provide an important foundation for future studies in understanding mechanisms that regulate the V(D)J recombinase during genotoxic stress. The ability to enhance such a regulatory process may lead to decreased risks of developing lymphoid malignancies following exposure to radiation and other DNA damaging drugs. PUBLIC HEALTH RELEVANCE: The V(D)J recombinase, consisting of the RAG1 and RAG2 proteins, is essential for development of the adaptive immune system; however, errors in V(D)J recombination can result in genomic instabilities leading to an increased risk of lymphoid malignancies. Preliminary new data indicate the V(D)J recombinase is sequestered from the genome upon exposure to DNA damaging agents in a possible attempt to reduce aberrant recombination reactions. The goal of this study is to determine the mechanistic basis for this effect, and if such a regulatory process leads to reduced V(D)J recombinase activity.